P. aeruginosa is an opportunistic pathogen and a leading cause of nosocomial infections, especially in a setting of epithelial cell injury and in immunocompromised patients. P. aeruginosa is also an important pathogen in patients with chronic obstructive pulmonary disorder, bronchiectasis, community acquired pneumonia, ventilator-associated pneumonia and cystic fibrosis. Recent studies suggest that viral and P. aeruginosa infections of the lung are synergistic: however, little is known about the underlying mechanisms whereby P. aeruginosa exacerbates viral infections. Accordingly, the long-term goal of our research is to elucidate the cellular and molecular mechanisms whereby P. aeruginosa causes lung disease and to elucidate the mechanism whereby P. aeruginosa exacerbates viral infections of the lung. Recently, we demonstrated that P. aeruginosa secretes a protein called Cif (CFTR Inhibitory Factor, originally named because of its ability to inhibit CFTR-mediated Cl secretion by airway cells). Cif is secreted by laboratory and clinical isolates of P. aeruginosa and inhibits the host immune response by down-regulating CFTR Cl secretion, which reduces mucociliary clearance, and by reducing the abundance of TAP1 (Transporter Associated with Antigen Presentation), which suppresses vial antigen presentation by class I MHC and the ability of cytotoxic T lymphocytes to clear viral infections. Cif is the first example of a protein secreted by a bacterium that down regulates viral antigen presentation by MHC class I. Preliminary data demonstrate that Cif inhibits the deubiquitination of CFTR and TAP1 in polarized human airway epithelial cells by facilitating G3BP1 inhibition of USP10, a ubiquitin-specific protease, thereby increasing the amount of ubiquitinated, and degraded, CFTR and TAP1. Thus, the three specific aims in this proposal will: (1) Test the hypothesis that Cif, by facilitating G3BP1 inhibition of USP10, reduces the deubiquitination of CFTR, thereby increasing the degradation of CFTR by lysosomes; (2) Test the hypothesis that Cif, by facilitating G3BP1 inhibition of USP10, inhibits the deubiquitination of TAP1, thereby targeting ubiquitinated TAP1 for degradation by the proteasome, and (3) Test the hypothesis that Cif, by reducing TAP1 levels, reduces MHC class I viral antigen presentation and cytotoxic T lymphocyte-mediated viral clearance. The studies proposed in this application will utilize a multi- disciplinary approach to examine host-pathogen interactions whereby the pathogen (P. aeruginosa) residing in the mucus layer overlying airway cells secretes a protein (Cif) that enters human airway epithelial cells and alters host immunity (i.e., mucociliary clearance and viral antigen presentation by MHC class I). Thus, the long- term goal of these studies is to elucidate how P. aeruginosa reduces the ability of the lung to clear infections and, ultimately, identify new therapeutic approaches to eliminate drug resistant P. aeruginosa infections. PUBLIC HEALTH RELEVANCE: Bacterial infections affect ~2 million people in the US annually, result in ~99,000 deaths and require ~$4.5 billion/year in health care. Many of these infections are caused by the bacterium Pseudomonas aeruginosa. The long-term goal of our research is to understand how Pseudomonas aeruginosa causes lung infections and enhances viral infections, and to identify new therapeutic approaches to eliminate drug resistant Pseudomonas aeruginosa infections.